TGF-2 signaling directs diverse cellular responses including differentiation, proliferation, and cell cycle arrest and through these responses plays a central role human development and disease. Activation of the TGF-2 receptor leads to phosphorylation of the transcription factors, Smad2 and Smad3 (Smad2/3). Once phosphorylated, Smad2/3 accumulates in the nucleus where they must interact with other transcription factors in order to bind DNA and regulate transcription. The mechanism by which activation of Smad2/3 directs such diverse responses remains unclear. Specific transcription factors have been reported to interact with Smad2/3 but these transcription factors are present in many different cell types and interact with Smad2/3 at only a few genes. Our preliminary data provides a clue to understand the diverse effects of TGF-2 signaling. We found that lineage-specific transcription factors may determine which genes Smad2/3 regulate. TGF-2 signaling is required to maintain human embryonic stem cells and is also required to direct embryonic stem cells to differentiate into endoderm, which will give rise to the cells of the liver, gut and pancreas. ES cells can be differentiated into hepatocytes, but low yields and contaminating cells are major hurdles to overcome in growing hepatocytes for human therapy. The goal of this project is to understand how TGF-2 signaling can direct endodermal differentiation and use this knowledge to improve our ability to grow hepatocytes for use in regenerative medicine. To achieve these goals the project will 1) identify the key transcription factors that co- occupy DNA sites with Smad2/3 in human ES cells, 2) identify key transcription factors that co-occupy DNA sites with Smad2/3 during endodermal differentiation and are required for endodermal differentiation, and 3) determine which factors directly interact with Smad2/3 to recruit them to bind DNA in embryonic stem cells and during endodermal differentiation. Identification of specific protein-protein interactions that determine how developing cells respond to TGF-2 signaling would provide potential targets to modulate TGF-2 signaling in during development and improve the ability to produce hepatocytes and pancreatic cells for therapy. This research will be performed by Dr. Mullen in the laboratory of Dr. Richard Young at the Whitehead Institute, a leader in the fields of stem cell biology, transcription and genomics. Dr. Mullen received his Ph.D. training in immunology and clinical training in gastroenterology. He is building on a background in molecular genetics and cell biology and a clinical interest in liver disease to develop a proposal to apply genomics and systems biology to understand the role of TGF-2 signaling in embryonic stem cells maintenance and endodermal differentiation. The Whitehead Institute is the ideal environment in which to perform this research given the collaborative atmosphere, facilities, educational opportunities and experience training successful physician scientist. Dr. Mullen will also work closely with his co-mentor at Massachusetts General Hospital, Dr. Ramnik Xavier who has expertise in genomics and application of systems biology to signaling pathways. In addition, Dr. Mullen has formed an advisory committee to support his research and his development into an independent investigator. The committee members are experts in stem cell biology, development and signal transduction and have extensive experience mentoring physician scientists. ! PUBLIC HEALTH RELEVANCE: TGF-2 signaling plays an essential role in human development and in many human diseases (1-4). The goal of this project is to learn how the TGF-2 signaling pathway can be required to regulate pluripotency in human embryonic stem (ES) cells yet also direct human ES cells to differentiate into endoderm, the precursor of cells of the liver and gut. Understanding which factors control this change in activity has direct application in regenerative medicine and the production of hepatocytes for therapy. !